TABLE 2.

Current methods for spatial transcriptomics

Method (reference)	RNA capture method	Labeling radius	Isolation time	Sample type	Application(s)
APEX2-seq (68)	In vitro RNA isolation and library prep	20 nm	1 h	Cell culture, in vivo labeling	Organelles, cells, in vivo transcriptomics
Considerations
Pros: excellent spatial resolution, labeling time, and read depth
Cons: requires genetic modification of “bait” protein; subcellular pools of bait protein cannot be resolved
TSA-seq (78)	In vitro RNA isolation and library prep	0.5 μm	24 h	Live/fixed cells and tissue	Organelles, cells, in situ transcriptomics
Considerations
Pros: good spatial resolution and excellent read depth; applicable to primary cells/tissues
Cons: subcellular pools of bait cannot be resolved; steric hindrance from antibody-enzyme
LCM (81, 83, 84, 89)	Image-targeted laser ablation/polymerization	10 μm	3 h	Live/fixed tissue	In situ cell proteomics
Considerations
Pros: user validates target region by imaging
Cons: low RNA yield/coverage; cannot resolve single cells borders; labor- and time-intensive
FISSEQ (99)	In situ rolling circle amplification and SOLiD	0.4 μm	48 h	Fresh/frozen/fixed	In situ cell transcriptomics
Considerations
Pros: user validates target region by imaging; good labeling radius with in situ cDNA synthesis
Cons: very low mRNA coverage; reliably reports only most abundant transcripts
10×/Visium (100)	On-slide primer array and cDNA amplification	55 μm	≥2 h	Fresh/frozen tissue	In situ cell transcriptomics
Considerations
Pros: user validates target region by imaging; commercial platform
Cons: low coverage confounded by weak spatial resolution (5–20 cells) of on-slide barcode array; expensive
Slide-seq (101)	On-slide bead array and SOLiD	20 μm	≥3 h	Fresh/frozen tissue	In situ cell transcriptomics
Considerations
Pros: reasonable spatial resolution (1–2 cells); 62% of beads in array map to 1 cell; cost-effective
Cons: no imaging; cell type position inferred from transcripts recovered; low mRNA coverage at 100–1,000 transcripts/bead
HDST (102)	On-slide bead array and cDNA amplification	13 μm	≥3 h	Fresh/frozen tissue	In situ cell transcriptomics
Considerations
Pros: good spatial resolution due to binning reads in hexagonal bead array (Lightseq > HDS > Slide-seq >> 10×/Visium); 86% of transcriptome identified across entire sample
Cons: no imaging; cell type position inferred from transcripts recovered; low mRNA coverage at 1.3% of transcripts/bead
Light-seq (103)	Light-directed barcoding and spatial indexing	2 μm	≥3 h	Fixed tissue	In situ cell transcriptomics
Considerations
Pros: good spatial resolution due to light-conjugated barcoding (Light-seq > HDS > Slide-seq >> 10×/Visium); pooling 25 similar cells yields 85% coverage of transcriptome ~3,500 transcripts
Cons: specialized microscopy and photolithography reagents needed